Factor XIII (also known as fibrin stabilizing factor, fibrinoligase, or plasma transglutaminase) is a plasma glycoprotein that circulates in blood as a zymogen (M.sub.r =.about.320 kD) complexed with fibrinogen (Greenberg and Shuman, J. Biol. Chem. 257: 609614 6101, 1982). Plasma factor XIII zymogen is a tetramer consisting of two a subunits (M.sub.r =.about.75 kD) and two b subunits (M.sub.r =.about.80 kD) (Chung et al., J. Biol. Chem. 249: 940-950, 1974) having an overall structure designated as a.sub.2 b.sub.2. The a subunit contains the catalytic site of the enzyme, while the b subunit is thought to stabilize the a subunit or to regulate the activation of factor XIII (Folk and Finlayson, Adv. Prot. Chem. 31: 1-133, 1977; Lorand et al., Biochem. Biophys. Res. Comm. 56: 914-922, 1974). The amino acid sequences of the a and b subunits are known (Ichinose et al., Biochemistry 25: 6900-6906, 1986; Ichinose et al., Biochemistry 25: 4633-4638, 1986). Factor XIII occurs in placenta and platelets as an a.sub.2 homodimer.
In vivo, activated factor XIII (factor XIIIa) catalyzes cross-linking reactions between other protein molecules. During the final stages of blood coagulation, thrombin converts factor XIII zymogen to an intermediate form (a'.sub.2 b.sub.2), which then dissociates in the presence of calcium ions to produce factor XIIIa, a homodimer of a' subunits. Placental factor XIII is activated upon cleavage by thrombin. Factor XIIIa is a transglutaminase that catalyzes the cross-linking of fibrin polymers through the formation of intermolecular .xi.(.delta.-glutamyl) lysine bonds, thereby increasing clot strength (Chen and Doolittle, Proc. Natl. Acad. Sci. USA 66: 472-479, 1970; Pisano et al., Ann. N.Y. Acad. Sci. 202: 98-113, 1972). This cross-linking reaction requires the presence of calcium ions (Lorand et al., Prog. Hemost. Throm. 5: 245-290, 1980; Folk and Finlayson, Adv. Prot. Chem. 31: 1-133, 1977). Factor XIIIa also catalyzes the cross-linking of the .delta.-chain of fibrin to .alpha..sub.2 -plasmin inhibitor and fibronectin, as well as the cross-linking of collagen and fibronectin, which may be related to wound healing (Sakata and Aoki, J. Clin. Invest. 65: 290-297, 1980; Mosher, J. Biol. Chem, 250: 6614-6621, 1975; Mosher and Chad, J. Clin. Invest. 64.: 781-787, 1979; Folk and Finlayson, ibid.; Lorand et al., ibid.). The covalent incorporation of .alpha..sub.2 -plasmin inhibitor into the fibrin network may increase the resistance of the clot to lysis (Lorand et al., ibid.).
Factor XIII deficiency results in "delayed bleeding," but does not affect primary hemostasis (Lorand et al., ibid.) Current treatment practices for patients having factor XIII deficiencies generally involve replacement therapy with plasma or plasma derivatives, or with a crude placental factor XIII concentrate (Lorand et al., ibid.; Forbisch et al., Dtsch. med. WochenSchr. 97: 449-502, 1972; Kuratsuji et al., Haemostasis 11: 229-234, 1982).
Factor XIII is also useful in treatment of patients with disorders in postoperative wound healing (Mishima et al., Chirurg 55: 803-808, 1984; Baer et al., Zentrabl. Chir. 105: 642-651, 1980), scleroderma (Delbarre et al., Lancet 2: 204, 1984; Guillevin et al., La Presse Medicale 14: 2327-2329, 1985; Guillevin et al., Pharmatherapeutica 4: 76-80, 1985; and Grivaux and Pieron, Rev. Pnemnol. Clin. 43: 102-103 1987), ulcerative colitis (Suzuki and Takamura, Throm. Haeostas, 58: 509, 1987), colitis pseudomembranous (Kuratsuji et al., Haemostasis 11: 229-234, 1982) and as a prophylactic of rebleeding in patients with subarachnoid hemmorhage (Henze et al., Thromb. Haemostas. 58: 513, 1987). Furthermore, Factor XIII has been used as a component of tissue adhesives (U.S. Pat. Nos. 4,414,976; 4,453,939; 4,377,572; 4,362,567; 4,298,598; 4,265,233 and U.K. Patent No. 2 102 811 B).
A number of purification schemes for factor XIII have been described. Chung and Folk (J. Biol. Chem. 247: 2798-2807, 1972) prepared factor XIII from platelet-concentrated plasma or from a fibrinogen preparation. Cooke and Holbrook (Biochem. J. 141: 79-84, 1974) describe the purification of factor XIII from the Cohn-I fraction. The method involves multiple ammonium sulfate precipitation steps and fractionation on DEAE cellulose chromatography to purify factor XIII from plasma. Skrzynia et al. (Blood 60: 1089-1095, 1985) purified the a subunit of factor XIII from a placental concentrate by chromatography and ammonium sulfate precipitation. Zwisler et al. (U.S. Pat. No. 3,904,751) and Bohn et al. (U.S. Pat. No. 3,931,399) describe multistep isolation procedures which rely on the use of diamino-ethoxy-acridine lactate to precipitate factor XIII. This precipitating agent would be an unacceptable contaminant in a therapeutic composition. Falke (U.S. Pat. No. 4,597,899) describes the isolation of factor XIII from an extract of placenta by alcohol precipitation.
Many of the previously described methods for purifying factor XIII have been directed to isolating it from plasma, serum, or fractions thereof. These starting materials are already enriched for factor XIII, and the contaminating proteins are generally well characterized and removable by known methods. Moreover, many of the known factor XIII-based therapeutic compositions are plasma fractions that have been enriched for factor XIII and contain other plasma proteins such as fibrinogen and fibronectin. Consequently, previously described purification or enrichment schemes are poorly suited to preparing highly purified factor XIII from heterogeneous starting materials, including crude cell lysates, where contaminating proteolytic activity may be high or unacceptable contaminants may be present. Furthermore, many of these methods were developed for laboratory-scale purification and are difficult to scale up for economical preparation of therapeutic quantities of factor XIII.
There is therefore a need in the art for simple, economical methods for purifying factor XIII. There is a further need in the art for purification methods that provide factor XIII preparations having low levels of factor XIIIa. Such methods should lend themselves to large-scale production of highly purified factor XIII from crude starting materials, such as lysates of recombinant cells. The present invention provides such methods, together with other, related advantages.